Inhibiting the growth of plant viruses



Patented June 21, 1960 INmITING THE GROWTH OF PLANT VIRUSES Walter A. Darlington, Dayton, Ohio, assignor to Monsanto Chemical Company, St. Louis, Mo., a corporation of Delaware No Drawing. Filed Apr. 21, 1958, Ser. No. 729,520

12 Claims. (Cl. 167-30) The invention relates to the inhibition of the growth of viruses which attack plants, and more particularly to chemotherapy as applied to the control of plant viruses.

Viruses have been defined as submicroscopic entities capable of being introduced into specific living cells and reproducing inside such cells only. This definition summarizes the chief characteristic of viruses-small size, infectivity, host specificity, and complete dependence on the integrity of the host cell. The intimate relationship between the virus and the host cell is the most distinguishing feature of the virus, and necessitates a more subtle approach to the chemotherapy of viruses than to that of other infectious agents, such as bacteria and fungi.

Viruses have long plagued mankind, but their existence was recognized first in 1892. It is now known that viruses afiiict plants, insects, bacteria and animals, and seem to be increasing in importance. This invention is, of course, limited to the treatment of virus diseases of plants.

There seems no doubt that plant virus diseases are more important now than they were even a decade ago, though the reasons for this are far from clear. Some of this increase is no doubt due to the introduction of viruses from one country to another in imported bulbs, tubers and plants. It is a difficult matter to prevent the entry of viruses by quarantine regulations, especially when no one, including the exporter, may be aware of the existence of the virus in question. The virus of tomato spotted Wilt is a good example of this. It was first described in Australia in 1915; about 15 years later it was identified at Cam-bridge, England, in an ornamental plant found in a Cardiff nursery. At the present time, its distribution is practically world-wide, having been passed easily from country to country in dahlia tubers and other vegetative organs of plants.

Although no intelligent estimate of the damages caused by plant viruses can be made, the problem is now being recognized as serious. Virus diseases are a serious problem in the farming of sugar beet, sugar cane, tomato, bean, cocao, orange, tobacco, potato, peach, strawberry, raspberry, broccoli, cabbage, hops and many flowers. In severe cases, e.g. with peach and sugar beet, virus diseases may force abandonment of large areas for the cultivation of a crop. In less severe cases, a virus disease may result in a decrease in yield and quality. It has been estimated that tobacco mosaic virus causes an annual loss of 40 million pounds of tobacco in the United States of America alone.

The great trade in seed potatoes amounting to half a million tons a year between England, Scotland and Ireland is based entirely on the prevalence of potato viruses and their aphis vectors in England. One potato virus alone, the ubiquitous potato virus which in the United States used to be called the healthy potato virus, is responsible for a loss of ten percent of the Worlds potato crop, while the losses of potatoes in Great Britain, due to virus infection, have been put at one million tons per year. The grower of sugar-beets, especially in East Anglia, is only too familiar with virus yellows, which, in an early season infection, may reduce sugar content by 50%.

The situation as regards the cabbage and broccoli crop becomes yearly more serious. There are two aphis-borne viruses which attack those plants causing them to be dwarfed and crinkled and prevent the formation of heart or curd.

Of flowering plants, possibly dahlias are the most serious affected by viruses and, being propagated by tubers, the situation resembles in many ways the disastrous one which results from growing potatoes in England from home-saved seed.

In the United States, in addition to most of the viruses found in England, there are several other important diseases such as curly-top of sugar beets and yellows of asters. Both the viruses causing these diseases have a wide host range and give rise to serious infections in many other crops.

In tropical regions there are many important virus diseases such as swollen-shoot of cocoa, phloem-necrosis of tea, rosette of ground-nuts and many others. A serious situation has arisen in the clove industry in Zanzibar Where the clove trees have been dying in large numbers. The disease which causes the sudden collapse of vigorous trees has been called the sudden death disease and by a process of elimination of all other possible causes is now considered to be due to a virus.

Of all these tropical virus diseases, however, by far the most serious is swollen shoot of the cocoa tree. In the Gold Coast this virus disease probably started about 1920, although it was not till 1936 that reports were received of the dying back of large numbers of trees. At first there seemed to be only a few areas affected, but soon reports were received from many areas that the disease was spreading, and 10,000 trees were known to be dying. Up to 1939, trees were dying at the rate of one million a year. The annual rate between 1939 and 1945 was five million and between 1945 and 1948 it rose to fifteen million. It is clear that the cocoa industry will not long be able to survive losses on that scale.

Although the losses to viruses are impressive, few control measures have been developed. Of the curative measures investigated, selected inactivation of a virus by the application of heat is generally not practical, and chemotherapy has been used very little. Control of insecttransmitted virus diseases by control of the insect vectors, although attractive, has not been too successful.

Other control measures of a negative nature are: destruction of infected plants (roguing); isolation from sources of infection; eradication of alternate hosts; use of healthy planting material; and the use of disease-resistant varieties. Although these practices are adequate in certain cases, none is generally applicable and all have definite limitations.

It is an object of this invention to provide a chemical. method of treating plants to inhibit the growth of viruses in the plants.

It is another object of this invention to provide a chemical method of treating tobacco plants to inhibit the growth of tobacco mosaic virus therein.

It is a further object of this invention to provide a chemical method of treating field pinto beans to inhibit the growth of tobacco ringspot virus therein.

These and other objects of the invention will become apparent as the detailed description of the invention proceeds.

The invention is the method of inhibiting the growth of viruses in plants, by treating the plants with at least a sufficient amount of a chemical compound to inhibit and salts thereof, e.g. the hydrochloride, the'nitrate, the sulfate, the dodecylbenzenesulfonic acid salt, etc. In the formula Ar is an aromatic hydrocarbon radical having from 6 to 12 carbon atoms, R and R" are selected from. the class consisting of hydrogen and aliphatic hydrocarbon radicals having from 1 to 18 carbon atoms, and Y is selected from the class consisting of hydroxyl and thiol radicals. Of the above compounds the preferred compounds are those in which the amine radical and the hydroxyl or thiol radical are positioned ortho to one another. The experimental worlg has indicated that these ortho compounds are the most active.

The growth-inhibiting chemical can be applied to th plants in a number of different ways, but it is preferred to apply it by spraying the plant foliage with a water solution or suspension of the chemical in at least a sufficient concentration to inhibit virus growth in the plant. The chemical compound can be applied to the plant by spraying, dipping in the case of potted plants, dusting with the chemical dispersed in an inert powder, or by other conventional means, and the chemical can even be applied indirectly to the plant by treating the soil whereby the chemical is absorbed by the plant through its roots system. An additional method of applying would be by suspending small particles of the chemical in a stream of air or other gas, and spraying the plant with this suspension. The chemical can even be sprinkled on the plants in undiluted liquid or powder form, ifde sired. In any event the invention does not lie in the particular method of treating the plant. If the chemical is applied in diluted form as will usually be desirable, it will be applied in as concentrated a solution as is readily handleable and which will not cause substantial phytotoxic damage to the plants being treated.

If the chemical is applied in water diluent and is soluble in water, of course, no dispersant will be necessary, although a wetting agent may still be desirable for maximum effectiveness. If the chemical is not very soluble in water an emulsifying agent may be required to keep it dispersed such as, e.g. alkylbenzenesulfonates, polyalkylene glycols, salts of sulfated long-chain alcohols, sorbitan fatty acid esters, etc., and other emulsifying agents which can be used are listed, e.g., in the US. Department of Agriculture Bulletin No. E607. The active chemicals of the invention can also be applied, dissolved or dispersed in organic solvents, e.g., liquid hydrocarbons, provided they are substantially nonphytotoxic tothe plants. If applied admixed with an inert pulverulent carrier, such carriers as e.g. talc, bentonite, kieselguhr, diatomaceous earth, etc. can be used.

. The following is a list of a number of the active cheminophenol, N-tridecyl-o-aminophenol, N-heptadecyl-o-ami- I nophenol, N-octadecyl-o-aminophenol, N,N-diethyl-oaminophenol, N-methyl-N-allyl-o-arninophenol, N-vinylo-aminophenol, N-(l-butenylyo-aminophenol, N-(Z-butenyl)-o-aminophenol, N-isobutenyl-o-aminophenol, N- (1 hexenyl) o aminophenol, N (1 octynyl) oaminophenol, N (1 dodecenyl) o aminophenol, N- (8-heptadecenyl)-o-aminophenol, N-(l,4-butadienyl)-oaminophenol, N-ethynyl-o-aminophenol, N-(1-propynyl)- o-aminophenol, N-(Z-propynyl)-o-aminophenol, N,N- dihexyl o aminophenol, phenol, N-(8,l1 heptadecadienyl)-o-aminophenol, etc. Of the number of compounds listed above the preferred compounds are the compounds in which the amino and hydroxy groups are associated in ortho arrangement, only one of R and R" is a hydrocarbon, and the hydrocarbon is either an unsaturated lower aliphatic hydrocarbon radical or a long-chain alkyl radical having from about 7 to 18 carbon atoms. It appears that the longer chain alkyl radicals enhance the activity of the compound but also that the shorter chain unsaturated aliphatic radicals enhance the activity of the compounds. Salts of the aminophenols are also active but the active compound is actually the aminophenol itself and not the salt portion. The aminophenol salts on contact with water hydrolyze releasing the aminophenol which is the active constituent. The salt in some cases can have the advantage of getting the aminophenol compound into the plant more efficiently and so promoting the anti-virus action. This can be particularly true of some of the organic salts such as one which will be mentioned illustratively below since the organic salts will tend to hydrolyze more slowly than the inorganic salts mentioned. The following is a non-limiting list of the active, salts of the invention: o-aminophenol-hydrochloride, p-aminophenol-sulfate, o-aminophenol-nitrate, N-allyl-o-aminophenol-dodecylbenzenesulfonic acid, etc.

An illustrative listing of the aminobenzenethiols is as follows: o-aminobenzenethiol, m -aminobenzenethiol, p-aminobenzenethiol, 3-mercapto-4-aminotoluene, 4-mercapto 5 amino-o-xylene, N ethyl o aminobenzenethiol, N,N-dimethyl-o-aminobenzenethiol, N-allyl-o-aminobenzenethiol, N-vinyl-p-aminobenzenethiol, N-dodecylo aminobenzenethiol, N (1 dodecenyl) o aminobenzenethiol, N (B heptadecenyl) o aminobenzenethiol, o-aminobenzenethiolhydrochloride, o-aminobenzenethiol-dodecylbenzenesulfonic acid, etc. a The following is an illustrative listing of the antivirus aminonaphthols of the invention: 2-aminonaphthol, 3-aminonaphthol, 4-aminonaphthol, S-aminonaphthol, 6-aminonaphthol, 7-aminonaphthol, S-aminonaphthol, aminonaphth-Z-ol, aminonaphth-B-ol, aminonaphth-G-ol, aminonaphth 7 ol, 4 methyl 2 aminonaphthol, 4,6 dimethyl 2 aminonaphtol, N allyl 2 aminonaphthol, N dodecyl 2 aminonaphthol, N,N dimethyl 2 aminonaphthol, N allyl 2 aminonaphthol, N-dodecyl-2-aminonaphthol, N,N-dimethyl-Z-aminonaphthol. N-ethynyl-Z aminonaphthol, .N-dodecyl-2- aminonaphtholhydrochloride, Z-aminonaphtholdodecylbenzenesulfonic acid, etc.

7 The following is an illustrative listing of the amino naphthalenethiols of the invention: 2-aminonaphthalene thiol, 3-aminonaphthalenethiol, 4-aminonaphtha1enethiol, 5 -aminonaphthalenethiol, 6-aminonaphthalenethiol, 7- aminonaphthalenethiol, S-aminonaphthalenethiol, aminonaphthalene-Z-thiol, aminonaphthalene- 3 -thiol, aminonaphthalene-61thiol, arnjnonaphthalene-7-thiol, 4-methy1- Z-aminonaphthalenethiol, 4,7-dimethyl-2-aminonaphthalenethiol, N-allyl-Z-aminonaphthalenethiol, N-dOde'cyl Z- aminonaphthalenethiol, N,N-dipropyl-Z-aminonaphthalenethiol, 'N-ethynyl 2 aminonaphthalenethiol, Z-amino naphthalenethiolhydrochloride,. N-dodmyl-Z-aminonaphthalenet-hiol-dodecylbenzenesulfonic acid, etc.

Active antivirus agents are also aminohydroxyphenyl compounds and an illustrative listing thereof is as follows: 2-amino-3-hydroxybiphenyl, 2-amino-4-hydroxybiphenyl, 3'-amino-4-hydroxybiphenyl, 3-amino-3-hydroxybiphenyl 4-ainino-3'-hydroxybiphenyl; N-al1yl-3-atnino-4- N,N didodecyl o aminohydroxybiphenyl, N-do'decyl-3-amino-4-hydroxybiphenyl, 3-amino-4-hydroxybiphenyl-hydrochloride, etc.

Aminohydroxy derivatives of other hydrocarbons falling within the broad definition of the invention will also be active antivirus agents, e.g. such hydrocarbons as indene, acenaphthene, fluorene, phenanthrene, and anthracene, etc. These derivatives are not considered as important, so a long listing of such compounds is not believed justified as unnecessarily lengthening the appli'cation.

The inhibiting effect of the chemicals on virus growth is illustrated as follows: In experiments run substantially according to the method of Commoner et al., Arch, Biochem. Biophys. 27, 271 (1950), tests were conducted showing the-inhibiting effects of-the chemicals against the multiplication of tobacco mosaic virus. Briefly the procedure involves inoculation of a young leaf of a healthy Turkish tobacco plant with the virus and, after 24 hours, contact of portions of the inoculated leaf with the test compound.

Leaves were inoculated, with Johnson'tobacco virus by rubbing their entire surface with a gauze pad moistened with a phosphate buffer solution (pH 7.0) containing 200 g. of the virus per milliliter. After inoculation the leaves were'placed under a bell-jar with their petioles in water for 24 hours. At the end of this time, six 0.5" discs were punched-from each leaf, weighed, washed in water and the discs of eachleafplaced in different 3.5" Petri dishes each containing a different test chemical in the desired concentration for testing in 15 ml. of halfstrength Vickerys solution (Vickery et al., Bull. Con. Agr. Expt. Sta., 399 (1937)), which had been prepared to have a final concentration of 5 X" mole of KH PO A control was prepared for each leaf by placing another set of six discs from the leaf into a Petri dish containing ml. of the same Vickerys solution but no test chemical. The dishes of discs were incubated for seven days under fluorescent light of 170 foot candles. At the end of that time the discs were removed and two groups of three were made up from each dish in order to pro vide checks. The tobacco mosaic virus content of each group was determined as described by Commoner et al. (loc. cit.) except that the final Washing step thereof was omitted because it had been previously observed by us that this step in the isolation procedure results in some loss of virus. The colorimetric measurements were made at 750 m in a Coleman Universal spectrophotometer and the amount of virus was read from a standard curve prepared with known amounts of tobacco mosaic virus.

Empolying this procedure inhibition of the virus growth was shown for the various test compounds at concentrations which are reported in the Table I below. Some of the more active compounds were tested at lower and lower concentrations in an attempt to determine their minimum effective concentration.

One of the more active compounds, namely, o-aminophenol-H01, was also subjected to additional testing on whole plants and the decrease in local lesions ob served on those plants treated with the chemical as compared to control plants which had no chemical treatment. The host plants used in testing the inhibition of tobacco mosaic (TMV) virus were tobacco plants (Nicotiana Glutinosa), and the host plants used in testing the inhibition of tobacco ringspot virus (TRV) were field pinto beans. A typical test is described in the following paragraph; however, a number of variations on this method have been tried such as, varying the time interval between inoculation and treatment, chemical treatment preceding inoculation, etc. Of course, a control is run with each test so the results in each case indicate the effectiveness of the compound.

Plants of the same size and age are divided into equal groups of controls and experimentals. The smaller and older leaves are removed from plants (only the primary leaves are used in the case of the bean plants) leaving only leaves of approximately equal size which are to be used in the experiment. These leaves are dusted lightly with Carborundum, then the leaves are inoculated 'by painting them lightly with a virus solution or an extract from a virus-infected plant. One hour later the experimental plants are sprayed with the test solutions which were aqueous solutions containingthe chemical in the desired concentration and about 1% of a nonionic surface active agent Atlox 1256 reputed to be a tall oil and ethylene oxide condensation product. The plants are then kept in the greenhouse for 3 to ,6 days during which time numerous discrete local lesions appear on the inoculated leaves. The lesions are counted and by comparison with the number of lesions on the control (untreated) plants, the effectiveness of the particular chemical in inhibiting the multiplication of the virus is determined. In the test on the tobacco plants using "a 10- molar concentration of the chemical, a 28% inhibition of the tobacco mosaic virus was observed. In the test on the bean plants using a 10- molar concentration of the chemical, a 14% inhibition of the tobacco ringspot virus was observed.

Thus, the data described above demonstrates the high and unexpected activity of the chemical compounds of the invention in inhibiting the growth of plant viruses. A number of different chemical compounds are shown to be active antivirus agents Tobacco mosaic virus is typical of the group of viruses of the mosaic type, and tobacco ringspot virus is typical of a second group of viruses of the necrotic type. These are the two main known types of virus diseases. It has been shown experimentally that both of these types of viruses are controlled by the inventive method, and since viruses are quite similar chemically it would be expected that viruses generally would be controlled by the method.

Although the invention has been described in terms of specified examples which are set forth in considerable detail, it should be understood that this is by way of illustration only and that the invention is not necessarily limited thereto, since alternative embodiments will become apparent to those skilled in the art in view of the disclosure. For example, the term hydrocarbon radical is used throughout the patent application in its broader sense, in that the R and R" groups or the aromatic radical can also contain substituents other than carbon and hydrogen, such as, halo, nitroso, nitro, sulfonic acid, carboxyl, hydroxyl, etc. which may even enhance to a degree the anti-virus activity of the compound, while in other cases the activity will be some what reduced. One skilled in the art will recognize that a compound containing a hydrocarbon radical that contains the substituents is the equivalent of the corresponding compound in which the hydrocarbon radical contains only carbon and hydrogen. Particularly, it should be recognized that the claimed compounds and the salts thereof are equivalent since a salt on contact with water will hydrolyze releasing the compound itself, which is the active anti-virus agent, so actually treating plants with the salt is in effect treating the plants with the compound itself. Accordingly, modifications are contemplated which can be made without departing from the spirit of the described invention.

.1 claim:; j a

1. The method of inhibiting the multiplication of plant virusescomprising applying to living plants a virus growthrinhibiting quantityof a compound of the for- R' -N- Ar Y wherein Ar is-an aromatic hydrocarbon radical having from 6 to 12 carbon atoms, R'- and R are selected from the class consisting of hydrogen and aliphatic hydrocarbon radicals having from 1 to 18 carbon atoms, and Y is selected from the class consisting of hydroxyl and thiol radicals.

2. The method of claim 1 wherein the amino and wherein Ar is an aromatic hydrocarbon radical having from to '12 carbon atoms.

6. The method of claim- 5 wherein said compound is o-aminophenol.

7 The method-of claim 5 wherein said compound is l-aminonaphth-Z-ol. e 8. The method of inhibiting the multiplication of plant viruses comprising applying to living plants a virus growth-inhibiting quantity of a compound of the formula 1 HgN-AP-SH wherein Ar is an aromatic hydrocarbon radical having from 6 to 12 carbon atoms.

9. The method of claim 8 wherein said compound is Z-aminobenzenethiol.

10. The method of inhibiting the multiplication'of plant viruses comprising applying to living plants a virus growth-inhibiting quantity of a compound of the forwherein Ar is an aromatic hydrocarbon radical having from 6. to 12 carbon atoms, and R is an aliphatichydrocarbon radical having from 1 to 18 carbon atoms.

11. The method of claim 10 wherein said compound is 2-(dodecylamino)phenol.

12. The method of claim 10 wherein said compound is Z-(allylamino) phenol. v 1

References Cited in the file of this patent King: U.S.D.A., Handbook No. 69, May 1954, pp. and 258. 

1. THE METHOD OF INHIBITING THE MULTIPLICATION OF PLANT VIRUSES COMPRISING APPLYING TO LIVING PLANTS A VIRUS GROWTH-INHIBITING QUANTITY OF A COMPOUND OF THE FORMULA 